Bottom Line:
Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates.The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa.This suggests that acRPB1 may have played a role in the success of animals.

Background: RPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes.

Results: To investigate the origins and function of RPB1 CTD acetylation (acRPB1), we computationally reconstructed the evolution of the CTD repeat sequence across eukaryotes and analyzed the evolution and function of genes dysregulated when acRPB1 is disrupted. Modeling the evolutionary dynamics of CTD repeat count and sequence content across diverse eukaryotes revealed an expansion of the CTD in the ancestors of Metazoa. The new CTD repeats introduced the potential for acRPB1 due to the appearance of distal repeats with lysine at position seven. This was followed by a further increase in the number of lysine-containing repeats in developmentally complex clades like Deuterostomia. Mouse genes enriched for acRPB1 occupancy at their promoters and genes with significant expression changes when acRPB1 is disrupted are enriched for several functions, such as growth factor response, gene regulation, cellular adhesion, and vascular development. Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates.

Conclusions: Our combined functional and evolutionary analyses show that RPB1 CTD acetylation was possible in the early history of animals, and that the K7 content of the CTD expanded in specific developmentally complex metazoan lineages. The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa. This suggests that acRPB1 may have played a role in the success of animals.

Fig5: Summary of the origins of subsets of genes regulated by acRPB1 with respect to relevant evolutionary events. The period between K7 repeat expansion and the diversification of vertebrates, shows particular enrichment for the origin of acRPB1 regulated genes and genes with functions relevant to animals. Each column in the heat map represents the evolutionary origin distribution of a set of genes. Color intensity reflects the magnitude of increase over background in each evolutionary window. Due to the large difference for some gene sets, two scales were necessary (red and purple). Asterisks indicate significant increase over background (Bonferroni-corrected p < 0.05). Relevant evolutionary events are identified with blue triangles. For reference, the first two columns represent the acRPB1 enriched and dysregulated origin distributions (Figure 4). The remaining columns give the origin distributions for functional subsets of genes enriched among acRPB1 sensitive genes.

Mentions:
Many genes involved in functions enriched among the acRPB1-regulated genes have origins close to the origin of K7 repeat-containing CTDs or later during the origin and radiation of vertebrates (Figure 5). The window between the K7 repeat expansion and the diversification of vertebrates is enriched with the origin of acRPB1-regulated genes. For example, the EGF-induced genes are significantly enriched for origins on the branch leading to Euteleostomi (p < 0.0001), as are the PDGF-induced genes (p < 0.0001) (Figure 5). Consistent with this pattern, twelve of the 17 (71%) genes in the EGF signal transduction pathway (BIOCARTA), which are necessary for EGF induction, were born between the origin of Bilateria and Euteleostomi; three were present in the last common ancestor of eukaryotes (e.g., MAPK), and only one appeared after the Euteleostomi (Additional file 6). Thus, this pathway’s evolutionary history mirrors that of the genes influenced by acRPB1 (Figure 4).Figure 5

Fig5: Summary of the origins of subsets of genes regulated by acRPB1 with respect to relevant evolutionary events. The period between K7 repeat expansion and the diversification of vertebrates, shows particular enrichment for the origin of acRPB1 regulated genes and genes with functions relevant to animals. Each column in the heat map represents the evolutionary origin distribution of a set of genes. Color intensity reflects the magnitude of increase over background in each evolutionary window. Due to the large difference for some gene sets, two scales were necessary (red and purple). Asterisks indicate significant increase over background (Bonferroni-corrected p < 0.05). Relevant evolutionary events are identified with blue triangles. For reference, the first two columns represent the acRPB1 enriched and dysregulated origin distributions (Figure 4). The remaining columns give the origin distributions for functional subsets of genes enriched among acRPB1 sensitive genes.

Mentions:
Many genes involved in functions enriched among the acRPB1-regulated genes have origins close to the origin of K7 repeat-containing CTDs or later during the origin and radiation of vertebrates (Figure 5). The window between the K7 repeat expansion and the diversification of vertebrates is enriched with the origin of acRPB1-regulated genes. For example, the EGF-induced genes are significantly enriched for origins on the branch leading to Euteleostomi (p < 0.0001), as are the PDGF-induced genes (p < 0.0001) (Figure 5). Consistent with this pattern, twelve of the 17 (71%) genes in the EGF signal transduction pathway (BIOCARTA), which are necessary for EGF induction, were born between the origin of Bilateria and Euteleostomi; three were present in the last common ancestor of eukaryotes (e.g., MAPK), and only one appeared after the Euteleostomi (Additional file 6). Thus, this pathway’s evolutionary history mirrors that of the genes influenced by acRPB1 (Figure 4).Figure 5

Bottom Line:
Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates.The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa.This suggests that acRPB1 may have played a role in the success of animals.

Background: RPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes.

Results: To investigate the origins and function of RPB1 CTD acetylation (acRPB1), we computationally reconstructed the evolution of the CTD repeat sequence across eukaryotes and analyzed the evolution and function of genes dysregulated when acRPB1 is disrupted. Modeling the evolutionary dynamics of CTD repeat count and sequence content across diverse eukaryotes revealed an expansion of the CTD in the ancestors of Metazoa. The new CTD repeats introduced the potential for acRPB1 due to the appearance of distal repeats with lysine at position seven. This was followed by a further increase in the number of lysine-containing repeats in developmentally complex clades like Deuterostomia. Mouse genes enriched for acRPB1 occupancy at their promoters and genes with significant expression changes when acRPB1 is disrupted are enriched for several functions, such as growth factor response, gene regulation, cellular adhesion, and vascular development. Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates.

Conclusions: Our combined functional and evolutionary analyses show that RPB1 CTD acetylation was possible in the early history of animals, and that the K7 content of the CTD expanded in specific developmentally complex metazoan lineages. The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa. This suggests that acRPB1 may have played a role in the success of animals.